Summary

The annexin A5 gene (Anxa5) was recently found to be expressed in
the developing and adult vascular system as well as the skeletal system. In
this paper, the expression of an Anxa5-lacZ fusion gene was
used to define the onset of expression in the vasculature and to characterize
these Anxa5-lacZ-expressing vasculature-associated cells. After
blastocyst implantation, Anxa5-lacZ-positive cells were
first detected in extra-embryonic tissues and in angioblast progenitors
forming the primary vascular plexus. Later, expression is highly restricted to
perivascular cells in most blood vessels resembling pericytes or vascular
smooth muscle cells. Viable Anxa5-lacZ+
perivascular cells were isolated from embryos as well as adult brain meninges
by specific staining with fluorescent X-gal substrates and cell-sorting. These
purified lacZ+ cells specifically express known markers of
pericytes, but also markers characteristic for stem cell populations. In vitro
and in vivo differentiation experiments show that this cell pool expresses
early markers of chondrogenesis, is capable of forming a calcified matrix and
differentiates into adipocytes. Hence, Anxa5 expression in perivascular cells
from mouse defines a novel population of cells with a distinct developmental
potential.

Introduction

Formation of the embryonic vascular system happens before nutrition by
diffusion runs into its limitation, and represents the first organ system in
the early embryo (Carmeliet,
2003; Noden,
1989). Vasculogenesis, the process of de novo formation of
vascular structures, is characterized by the differentiation of mesodermal
derived angioblast precursor cells into endothelial cells forming the primary
capillary plexus (Risau, 1997;
Risau and Flamme, 1995).
Subsequently, during angiogenesis this early vascular system undergoes a
period of pruning, remodeling and maturation. Additionally, it takes two cell
types to generate functional blood vessels, endothelial cells and perivascular
cells (Gerhardt and Betsholtz,
2003). Perivascular cells (PVC) can be classified as pericytes in
capillaries or vascular smooth muscle cells (vSMC) in larger vessels,
depending on their morphology and location. Both may represent subtypes of a
continuum of related cell types.

PVCs are essential for the development of functional vessel walls and
contribute to the structural integrity and contractility of vessels. However,
developmental origins of PVCs remain unclear. Different speculations were
proposed, such as derivation from mesenchymal and epicardial cells or neural
crest (Gerhardt and Betsholtz,
2003), but also transdifferentiation of endothelial cells is
discussed (DeRuiter et al.,
1997). Recent data support the idea of a common progenitor of
perivascular and endothelial cells – the FLK1-positive angioblast
(Ema et al., 2003;
Yamashita et al., 2000).

Previous studies have shown that PVCs can differentiate into various cell
types such as adipocytes, chondrocytes, fibroblasts and macrophages
(Canfield et al., 2000;
Diaz-Flores et al., 1992).
These cells may therefore reflect in some aspects the phenotype of mesenchymal
stem cells (MSC) originally isolated from bone marrow stroma
(Caplan, 1991). Owing to
general difficulties with isolation and characterization of pericytes, it is
not yet possible to purify these cells from mouse. Therefore, detailed
analysis of pericytes and PVCs in the mouse system is still pending.

Annexin A5 represents a typical member of the annexin family, characterized
by the ability to bind to phospholipid membranes in the presence of
Ca2+ (Moss et al.,
1991). Although numerous functions of annexin A5 have been
described in vitro, its in vivo role still remains unclear
(Brachvogel et al., 2003).
Expression analysis of the annexin A5 gene (Anxa5) by the use of an
Anxa5-lacZ fusion gene in vivo showed that this gene is
expressed in vasculature-associated cells and at later stages (E13.5-P1) in
mesenchymal condensations, chondrocytes as well as all skeletal elements
(Brachvogel et al., 2001).

In this report, we used the Anxa5-lacZ promoter trap
first to describe the onset of Anxa5-lacZ expression early
in development and second to characterize the
Anxa5-lacZ+ cells of different developmental
stages in vitro and in vivo. Surprisingly, Anxa5-lacZ
expression is initially detected in cells associated with the primary vascular
plexus. Later, expression is restricted to cells associated with endothelial
cells. By using fluorescence-activated cell sorting (FACS) we were able to
purify and characterize Anxa5-lacZ+ PVCs for the
first time from mouse. The purified cell populations revealed unique
expression profiles of markers characteristic for pericytes and mesenchymal
stem cells. Additionally, these isolated PVCs have a capacity for
differentiation into mesenchymal cell lineages. In the future, this will
enable us to define the developmental ontogeny and plasticity of the
heterogeneous, poorly characterized perivascular cell population.

Materials and methods

Mouse strains

Ablation of the annexin A5 gene by homologous recombination and generation
of an Anxa5-lacZ fusion gene has been described recently.
Genotyping of mice was carried out as described
(Brachvogel et al., 2003).
Experiments were performed with mice displaying mixed genetic background of
C57/BL6x129/SvJ. Immunodeficient NOD/SCID mice were purchased from Taconic
M&B, Denmark.

Detection of β-galactosidase activity

Whole-mount staining of embryos was performed after fixation in 0.2%
glutaraldehyde for 10 minutes and stained in X-gal solution at room
temperature for 24 hours. Treated embryos were dehydrated by increasing
concentrations of ethanol and embedded in paraffin wax according to standard
procedures. Embryos were cut into 9 μm sections (Leica RM 205), dried for 1
hour at 60°C and de-waxed in xylene. Specimens were counterstained in
eosin for morphological assessment.

Purification of lacZ-expressing cells

Cells were isolated from heterozygous embryos of different stages
(E8.5-E16.5) or adult brain meninges (dura, arachnoid and pia mater). In
embryos, the heart was removed. Residual embryos and meninges were digested
with 0.4% collagenase II (Worthington) for 60 minutes at 37°C, followed by
2% trypsin (Sigma)/100 U DNAse I (Roche) for 30 minutes at 37°C.
Subsequently, cell suspensions were filtered through a 100 μm nylon mesh
(Becton Dickinson). Suspensions were stained for β-galactosidase activity
as described (Miles et al.,
1997). Briefly, 1 ×106 cells were suspended in 20μ
l PBS and added to 20 μl of 2 mM
fluorescein-di-(β-D-galactopyranoside) FDG (Sigma). Cells were incubated
at 37°C for 75 seconds and subsequently 500 μl of ice-cold PBS were
added. Cells were stored on ice for 3 hours. The suspension was stained with
propidium iodide (PI, 1 μg/ml) to label dead cells during flow cytometry.
lacZ+ cells were detected by the FITC channel, besides
FSC/SSC and PI. Cells were sorted by fluorescence-activated cell sorting
(MoFlow, Cytomation) and viable PI–/lacZ+
cells were collected. Cells derived from wild-type embryos or adult brain
meninges were used as negative control.

Regeneration of muscle after crush injury

Regeneration studies of tibialis anterior muscle (TA) were carried out as
described (Mitchell et al.,
1992). Briefly, mice (15-20 weeks old) were anesthetized and a
transversal crush injury was induced. Immediately after damaging the muscle,
25 μl of FDG-sorted PVCs from meninges of 2-month-old mice (5×
104 cells/25 μl) were injected into damaged region with a
25 μl syringe. The wound was closed with 6-0 suture-silk; the mice were
allowed to recover in a warmed environment and placed in standard cages. Mice
were sacrificed 13 days after injury and the treated muscle was processed for
frozen sections.

Regeneration of bone marrow by
Anxa5-lacZ+ PVCs

The Anxa5-lacZ+ mutant was generated on a
C57BL/6 ×129SvJ background, displaying the Ly5.2 marker on the surface
of bone marrow cells (BMCs). lacZ+ PVCs of adult meninges
were isolated from heterozygous animals (2-4 months of age). Supporting BMCs
were isolated from C57BL/6-Ly-5.1 mice. Supporting BMCs (9×
105 cells) were mixed with
Anxa5-lacZ-positive PVCs (1 ×105 cells) and
injected (100 μl) into the tail vein of C57BL/6-Ly-5.1 recipient, that had
been given an irradiation dose of 900 Rad
(Kawada and Ogawa, 2001). As
control, BMCs from Anxa5-lacZ+-Ly-5.2 were mixed
with supporting BMCs from C57BL/6-Ly-5.1 and injected into irradiated
C57BL/6-Ly-5.1 recipients. Peripheral blood was isolated 4 weeks after
transplantation from the retro-orbital plexus of anaesthetized recipients and
C57BL/6-Ly-5.1 mice. Cells (106) were stained with PE-conjugated
Ly5.1 or FITC-conjugated Ly5.2 antibodies (Pharmingen) and incubated on ice
for 30 minutes. Cells were washed three times in PBS/5% FCS and stained for
dead cells with PI. Analysis was performed by flow cytometry detecting PE- and
FITC-specific fluorescence.

Results

Anxa5 is expressed during early stages of vasculogenesis

An Anxa5-lacZ promoter trap mutant was previously
generated to inactivate the murine annexin A5 gene (Anxa5) in mice.
Initial studies showed that this fusion gene is expressed in cells associated
with the vascular network of embryos (E10.5-E14.5)
(Brachvogel et al., 2001). To
define the onset of expression during development, we analyzed
Anxa5-lacZ+ expression during early embryogenesis
(E5.5-E9.5). The first expression of Anxa5-lacZ+ was
detected in the ectoplacental cone of embryos at E5.5, persisting during
development (Fig. 1). As soon
as vasculogenesis starts (∼E7.5), lacZ-activity was detected in
the developing capillary network of the yolk sac and dorsal aorta
(Fig. 1B). At E8.5 the
intensity of staining increased and all elements of the primary capillary
plexus in the yolk sac were stained (Fig.
1C,D). Interestingly, no staining was observed in the endoderm of
the yolk sac and in hematopoietic precursors of blood islands
(Fig. 1D,E). The lacZ
staining was clearly restricted to lining angioblasts of yolk sac
mesoderm.

At E9.5, after initial remodeling of the vascular plexus, the reporter gene
was still active in blood vessels of yolk sac
(Fig. 1F-I). Additionally,
staining of the intra-embryonic vasculature persisted at this time point and
again, only cells closely associated with the dorsal aorta and intersegmental
vessels express Anxa5-lacZ
(Fig. 1H,I). No blood cells
were stained. Therefore, the onset and persistence of
Anxa5-lacZ expression reflect the formation and development
of primary vasculature in yolk sac and embryo.

Anxa5-lacZ reporter is specifically expressed in the
vasculature of E10.5 embryos. Sagittal sections (A-D,F-I) of heterozygous
embryos were stained for β-galactosidase activity (A,F) followed by
immunodetection of SMA (B,G), PECAM (C,H) and nuclear staining by DAPI (D,I).
In the overlays (E,J), X-gal deposits are indicated by white spots.
Anxa5-lacZ+ cells are restricted to
SMA+/PECAM+ dorsal aorta and PECAM+
capillaries (E). At higher magnification, only a subset of PECAM-associated
cells was positive for lacZ, as shown by DAPI staining (J, arrows).
(K-N) Anxa5-lacZ+ cells are located in regions
positive for the pericyte-specific marker NG2-proteoglycan in embryonic brain
at E13.5. Transverse sections of capillaries were stained forβ
-galactosidase activity (K, arrows). Positive areas appear black because
of the monochrome character of the camera system. In parallel, PECAM (L) and
NG2-proteoglycan (M) were detected by immunostaining. After merging,
colocalization of PECAM and NG2 appears orange (N). X-gal deposits are shown
as white spots (N, arrows). Scale bar: 50 μm.

Anxa5-lacZ expression defines a population of perivascular
cells

Vascular elements expand and remodel during angiogenesis. At this stage,
vasculature is characterized by differentiated endothelial cells as well as
perivascular cells (PVCs). Initially, PVCs are loosely attached to the
endothelial network and not separated by a common basement membrane. To define
the location of Anxa5-lacZ expression, we combined the
detection of the β-galactosidase activity (β-gal) with specific
immunostaining for platelet endothelial cell adhesion molecule 1 (PECAM/CD31)
and smooth muscle actin (SMA) antibodies
(Fig. 2). Whereas PECAM marks
specifically the endothelial layer, SMA detects most vSMCs and pericytes
(DeRuiter et al., 1997). In
E10.5 embryos, it is clearly visible that lacZ-reporter expression
colocalizes with the expression of SMA and PECAM in the dorsal aorta
(Fig. 2A-E). Additionally,
smaller vessels expressing Anxa5-lacZ stained for PECAM, but
were negative for SMA (Fig.
2E). Labeling of nuclei by DAPI staining in the region of
intersegmental vessels revealed that only a subset of cells associated with
PECAM-positive endothelial cells are expressing Anxa5-lacZ
(Fig. 2F-J). This is also
indicated in capillaries of heterozygous E10.5 embryos costained with a
PECAM-specific antibody and X-gal (see Fig.
3A). By merging phase contrast and fluorescence images, it became
obvious that PECAM+ cells covered the inside lumen of the
capillary, whereas only some cells adjacent to the endothelial layer were
positive for Anxa5-lacZ (black arrows).
Anxa5-lacZ-expression was also seen in small capillaries of
the brain from heterozygous embryos at E13.5
(Fig. 2K-N). In transverse
sections, the deposits of the X-gal product are found in cells next to the
endothelial cells of capillaries in colocalization with NG2-proteoglycan, as
indicated by staining with specific antibodies
(Fig. 2M). The vessel character
was confirmed by immunostaining for PECAM
(Fig. 2L). These data prove
that only perivascular cells express the Anxa5-lacZ marker
at E10.5 and E13.5; lining endothelial cells do not.

Owing to the variable expression of markers in PVCs, their specificity
varies depending on the tissue- and development-related context
(Gerhardt and Betsholtz,
2003). Therefore, staining for SMA does not represent an
unequivocal marker for PVCs and their precursors. By contrast, electron
microscopy allows the identification of cells by their localization within
vessel structure. Specific staining for β-gal activity in
Anxa5-lacZ-expressing cells results in the formation of an
unusual cytoplasmatic deposit of the reaction product in expressing cells,
represented by an intracytoplasmatic vesicle of unknown origin (data not
shown). Hence, positive cells can be identified in electron microcopy by the
presence of these electron-dense vesicles
(Fig. 3B, arrow). In
heterozygous lacZ-expressing embryos of stage E10.5 these deposits
were exclusively detected in PVCs closely associated with capillaries, but not
in endothelial cells. The characteristic common basement membrane of
endothelial cells and pericytes of functional blood vessels is not yet visible
at these stages and therefore cannot be used for identification. These data
support the idea that Anxa5-lacZ represents a novel,
specific marker for PVCs.

Anxa5-lacZ+ cells are found associated with,
but are not identical to, the endothelium in capillaries of E10.5 embryos and
adult vasculature. (A) Sagittal sections of heterozygous embryos at E10.5 were
stained for lacZ activity and immunostained for PECAM. After merging,
Anxa5-lacZ+ regions appear red (arrows).
Anxa5-lacZ+ cells are located parallel to the
lining endothelial cells. (B) Ultrastuctural localization of
Anxa5-lacZ+ expression in heterozygous embryos at
E10.5 by electron microscopy. An electrondense deposit of X-gal product is
detected (arrow) exclusively in PVCs. (C,D) Sections from adult kidney tested
for lacZ activity (C) and SMA expression (D) showed colocalization in
the vascular walls of large blood vessels. The relative intensity of
fluorescence was reduced by X-gal staining. (E) Expression of the
Anxa5-lacZ reporter in brain of adult mice is found in the
pial vasculature. (F) X-gal staining (arrow) is restricted to cytoplasmic
vesicles in pericytes, characterized by the common basement membrane
(arrowhead) with endothelial cells. Capillaries (C), endothelial cells (EC)
and pericytes (P) are marked (B,F). Scale bars: 50 μm (A,C); 1 mm (E).

Similarly, Anxa5-lacZ+ was also expressed in
PVCs of adult mice as high levels of β-gal-activity were detected in
smooth muscle layers of larger vessels in many tissues such as muscle, lung,
brain and kidney. As an example, kidney was stained for β-gal activity
and with a SMA-specific antibody (Fig.
3C,D). Obviously, both stains colocalize and vSMCs express the
Anxa5-lacZ reporter. Pial vasculature of adult brain was
strongly positive for β-galactosidase, as shown in whole mount X-gal
staining (Fig. 3E). Electron
microscopy was used to localize the Anxa5-lacZ+
cells in the brain capillaries. Unique electron-dense staining aggregates were
only found within pericytes, which are identified in adult tissues by their
common basement membrane with the underlying endothelial cell
(Fig. 3F).

Detection and purification of lacZ+ cells by
fluorescence-activated cell sorting. Single cell suspensions of heterozygous
embryos at E12.5 were stained with the fluorescent lacZ substrate FDG
and cell death was detected by adding propidium iodide (PI). In FACS analysis,
only vital cells in gate R1 (FSC/SSC) were considered for data analysis (A).
Cells from wild-type embryos were used as control and almost no events were
detected in gate R2 (B). About 3% of the heterozygous cells analyzed,
represent vital Anxa5-lacZ+ cells (C). Similar
amounts of Anxa5-lacZ+ cells were detected in
embryos at E8.5 (D) and E10.5 (E) as well as in adult brain meninges (F). The
percentages of cells within the gate are depicted, genotypes (wild type, wt;
heterozygous, +/–) are indicated.

Pericytes had been isolated from various organs (brain, retina and lung) of
only some species but not from murine tissues yet
(Doherty and Canfield, 1999;
Schor and Canfield, 1998).
Therefore, we used the expression of the Anxa5-lacZ reporter
for detection and purification of these cells by cell sorting
(Fig. 4). Initially,
heterozygous embryos at stage E12.5 were used, which express the
Anxa5-lacZ reporter only in vasculature and heart
(Brachvogel et al., 2001).
After removal of the heart, Anxa5-lacZ+ cells
exclusively represent cells closely associated with vascular elements. After
dissociation of embryos, viable cells were stained in vivo with the
lacZ substrate fluoresceindi-β-D-galactopyranoside (FDG)
(Miles et al., 1997). Damaged
cells were excluded by propidium iodide (PI) staining. FDG-positive cells were
virtually absent in wild-type controls
(Fig. 4B). By contrast, a
population of cells exhibiting a strong fluorescent signal
(FDG+/PI–) was observed in heterozygous embryos
(Fig. 4C).

To test the limits of the method, we also isolated FDG+ cells
from E10.5 and E8.5 embryos. Embryos at E10.5 were similarly processed and
Anxa5-lacZ+ cells (2.5%) could be purified from
individual embryos (Fig. 4E).
By contrast, pools of heterozygous E8.5 embryos had to be used because of the
lower total cell numbers (Fig.
4D). Out of five heterozygous embryos (E8.5), only 15,000 cells
were isolated with a small subset (∼300 cells) representing
Anxa5-lacZ+ cells. Typically, isolations of cells
from different embryonic stages (E8.5-E12.5) resulted in a relative yield of
2-4% viable Anxa5-lacZ+ cells.

This experimental procedure could also be used for isolation of
Anxa5-lacZ+ cells from adult mice
(Fig. 4F). Vasculature of the
meninges is easily accessible and represents an appropriate source of
vascular-associated cells because of the low complexity of this tissue.
Therefore, single cell suspensions of isolated meninges of the dura, arachnoid
and pia mater were pooled from several
Anxa5-lacZ+ mice, stained with FDG and sorted forβ
-gal activity. About 1% of cells were positive and could be sorted from
the pool of vital cells. Typically, about 7000
Anxa5-lacZ+ cells can be isolated from the
meninges of an individual mouse. Depending on the gating stringency, an
enrichment of up to 99% lacZ-expressing cells was achieved after
sorting.

The purification of Anxa5-lacZ+ cells allowed
the analysis of the expression profile of these murine PVCs by qualitative
RT-PCR (Fig. 5). Sorted cells
from whole embryos at E10.5, isolated brains of E16.5 embryos and brain
meninges of adult animals were used for RNA isolation. Total RNA from E10.5
embryos represented the positive control. These RNAs were tested by RT-PCR for
the expression of NG2 and PDGFRβ, which represent characteristic markers
for pericytes (Gerhardt and Betsholtz,
2003). Purified PVCs were clearly positive for these markers. By
contrast, markers for muscle satellite stem cells (Pax7) or differentiating
muscle cells (Myod1) were not expressed. Therefore, purified
Anxa5-lacZ+ cells display typical markers of
vascular pericytes.

Anxa5-lacZ+ cells may represent a stem cell-like
population

Surprisingly, sorted cells from E10.5, E16.5 and adult meninges also
expressed the stem cell markers Flk1, Kit, Sca1, CD34 and low amounts of
VE-cadherin (Fig. 5A-C). None
of the sorted cells produced CD45 mRNA, a marker common to hematopoietic cells
(Asakura et al., 2002). The
expression profiles of sorted lacZ+ cells from various
developmental stages and tissues were highly homologous
(Fig. 5). A reproducible,
quantitative difference was seen for the relative expression of Sca1 and Kit,
with Kit being strongly expressed at E10.5 and E16.5, whereas only low levels
were seen in adult PVCs. By contrast, Sca1 was highly expressed in adult PVCs
but was detected at low amounts in purified cells from embryos (E10.5, E16.5).
These data indicate that the Anxa5-lacZ marker defines a
homogenous pool of cells from embryonic and adult vasculature, which resembles
characteristics of pericytes as well as stem cell populations
(Minasi et al., 2002).

Expression profiles indicated that isolated
Anxa5-lacZ+ PVCs may represent a pool of cells
with stem cell-like character. For testing their potential differentiation
capabilities isolated cells were cultured in vitro. Single PVCs displayed a
stellate morphology, as it was described for pericytes
(Canfield et al., 2000).
Subsequently, PVCs started to align and after reaching confluence, the
formation of multilayered areas is observed (data not shown).

Expression profiles of isolated Anxa5-lacZ+
cells. Cells were sorted from heterozygous embryos at E10.5 (A), dissected
brains from E16.5 embryos (B), adult brain meninges (C) and RNA was isolated.
RNA isolated from total embryos at E10.5 represented the positive control (D).
RT-PCR was used to detect the mRNA of specific markers for stem cells (Flk1,
Kit, Sca1, CD45, CD34, VE-cadherin), myogenic cells (Myod1) as well as
myogenic satellite cells (Pax7) and pericytes (PDGFRβ, NG2).
Anxa5 was tested as a positive control and reactions without mRNA (E)
were used as negative controls. GAPDH was used for standardization.

Owing to the ability of isolated bovine pericytes to differentiate into a
bone and cartilage-like phenotype (Canfield
et al., 2000), confluent layer of PVCs were tested for their
capability to differentiate into chondrogenic and osteogenic lineages. In
vitro chondrogenesis or osteogenesis was induced by culturing 2×
105 cells in three dimensional aggregates in the presence of
human BMP2 (Johnstone et al.,
1998) or ascorbate-2-phosphate
(Bianco et al., 1998),
respectively. Aggregates were harvested after 14 or 21 days of culture and
sections were immunostained for the presence of chondrogenic and
osteogenic-specific markers, reflecting various stages of differentiation,
such as collagen I, collagen II, collagen VI, collagen IX and collagen X
(Kong et al., 1993;
Quarto et al., 1993;
Zhang et al., 2003;
Zhou et al., 1995).
Differentiation to an early chondrogenic phenotype became evident after 14
days as the expression of collagen VI (Fig.
6C,F) as well as of collagen IX
(Fig. 6B,E) was clearly
detected. By contrast, collagen II as a marker for differentiated chondrocytes
was barely detectable even after 21 days in culture when compared with the
secondary antibody control (Fig.
6A,D). No signal was found for collagen X as a marker of
hypertrophic chondrocytes (not shown). Therefore, isolated PVCs have at least
the capacity to undergo some initial steps of differentiation towards
chondrogenic cells but do not develop a full chondrocyte phenotype.
Three-dimensional aggregates also respond to osteogenic signals in the medium,
as indicated by their brittle phenotype after 21 days in culture.
Subsequently, sections of these cultures were analyzed for Ca2+
deposits by van Kossa and Alizarin Red staining. The accumulation of
Ca2+ deposits was clearly detectable with both methods
(Fig. 6H,I), whereas no
staining was observed in aggregate cultures kept in proliferating medium
(Fig. 6K,L). Additionally,
aggregates cultivated in osteogenic medium expressed collagen I, as shown by
immunostaining on sections of these cultures
(Fig. 6G,J). Hence, PVCs can
organize a mineralizing bone-like matrix in vitro, as shown previously for
chondrocytes (Bianco et al.,
1998).

Mesenchymal stem cells are capable of differentiating into adipocytes; this
is the least understood pathway of mesenchymal stem cells differentiation
(Caplan and Bruder, 2001).
Confluent layers of isolated PVCs have therefore been cultivated in medium
that induces adipogenesis in mesenchymal stem cells or adult
trabecular-derived bone cells (Nuttall et
al., 1998), but as yet, no increased expression of specific
adipogenic markers, such as lipoprotein lipase (LPL) and
proliferator-activated receptor γ2 (PPARγ2), has been detected by
RT-PCR. Additionally, no excessive deposition of lipid droplets can be seen
using Oil red O staining (Nuttall et al.,
1998) (data not shown). Surprisingly, a differentiation into
adipocyte-like cells is detectable in aggregates cultured in chondrogenic
medium. A strong staining with Oil Red O, which is indicative of synthesis of
neutral lipids, is detectable on sections of these aggregate cultures
(Fig. 6M). This differentiation
event was never observed in cells cultured in confluent monolayer cultures.
Additionally, these cells express the adipocyte-specific protein PPARγ2,
as shown by immunostaining (Fig.
6N,O) (Chawla et al.,
1994). Therefore, we conclude that the cultured
Anxa5-lacZ+ population retain the potential to
differentiate into early chondrogenic, osteogenic and adipogenic cells in
three-dimensional aggregate cultures. A similar differentiation capacity has
been seen previously with isolated bovine pericytes
(Canfield et al., 2000).

Anxa5-lacZ+ cells differentiate into early
chondrogenic, osteogenic and adipocyte lineages in vitro. Aggregate cultures
of PVCs were cultivated in chondrogenic medium (A-F,M-O). By day 14
(B,C,E,F,M-O) or 21 (A,D), cryosections were performed and strong staining was
seen for collagen VI (Col6; C) and collagen IX (Col9; B), whereas collagen II
was barely detectable (Col2; A). Aggregate sections were stained for neutral
fat deposits using oil Red O staining (red droplets) and Hematoxylin (M) as
well as for PPARγ2 (N). Additionally, isolated PVCs were cultivated in
osteogenic medium for 21 days (G-L). Ca2+ deposits in cryosections
of these aggregates were detected by Alizarin Red (H) as well as van Kossa
staining (I). Collagen I was detected by immunostaining (G). Aggregates of
PVCs were cultivated in proliferating medium as negative control for the
staining of Ca2+ deposits (K,L). Secondary antibodies were used as
negative control for the immunostaining (D-F,J,O). Scale bars: 50 μm.

Isolated Anxa5-lacZ+cells express typical stem
cell markers and differentiate into adipocytes, chondrogenic and osteogenic
cells in vitro. To examine their developmental plasticity in vivo, we tested
their ability to participate in regeneration processes of skeletal muscle
(Asakura et al., 2002). As the
expression profiles of purified Anxa5-lacZ+ cells
from embryos and adult meninges were similar, we used sorted PVCs from adult
meninges. Sorted cells (50,000) were injected into regenerating tibialis
anterior (TA) muscle of immunodeficient NOD/SCID mice
(Shultz et al., 1995)
immediately after a local damage was induced into the muscle by crushing
(Mitchell et al., 1992). The
muscle was allowed to regenerate for 13 days, isolated and serial cryosections
were analyzed by staining with X-gal to track incorporated
Anxa5-lacZ+ cells. Isolated PVCs from adult
meninges were detectable after 13 days within the zone of regeneration,
whereas no stained cells were observable outside the regenerating zone
(Fig. 7). At day 13, most of
the meninges-derived PVCs were detected in columnar arrangements surrounding
regenerated desmin-positive muscle fibers but were excluded from intact or
regenerated muscle fibers (Fig.
7B,E). Interestingly, the injected cells were concentrated in
areas with high levels of CD34 expression that were not congruent with the
PECAM-positive vasculature (Fig.
7C,F). It is assumed that hematopoietic stem cells also express
CD34 (Dell'Agnola et al.,
2002; Mahmud et al.,
2002; Uchida et al.,
2001). In addition, some of the injected PVCs could be found in
regions of Sca1-expressing cells (not shown), another stem cell marker
(Asakura et al., 2002;
Howell et al., 2002). These
data indicate that injected Anxa5-lacZ+ PVCs are
retained and may participate in the regeneration process. Nevertheless,
localization does not implicate participation, and future studies will clarify
whether these cells have the potential to differentiate into muscle
fibers.

Anxa5-lacZ+ perivascular cells do not exhibit
hematopoietic potential

Adult stem cells have been characterized in many tissues and some
populations have revealed the potential for hematopoietic differentiation
(De Angelis et al., 1999;
Jackson et al., 2002).
Consequently, we addressed the issue of whether some of these
Anxa5-lacZ+ cells exhibit a capacity for
reconstitution of the hematopoietic system, as has been shown for murine
muscle fractions (Howell et al.,
2002). Therefore, we injected
Anxa5-lacZ+ PVCs from adult meninges into
lethally irradiated C57BL/6 mice, which expressed the polymorphic allele Ly5.1
on all nucleated hematopoietic cells (Shen
et al., 1986). Isolated PVCs (1 ×105) from 14
adult brain meninges of heterozygous, Ly5.2 expressing mice were mixed with
bone marrow cells (9 ×105 cells) from C57BL/6-Ly5.1 mice to
support the regeneration of the bone marrow. This mixture was injected into
the tail vein of lethally irradiated Ly5.1 recipient mice. As a positive
control, 1 ×105 bone marrow cells from Ly5.2-positive mice
were injected in a mixture with 9 ×105 bone marrow cells from
Ly5.1 mice. Four weeks after injection, Ly5.1 and Ly5.2 expression was
analyzed in the peripheral blood of bone marrow reconstituted mice
(Fig. 8). As expected, no
Ly5.2+ cells were detectable in untreated C57BL/6-Ly5.1 mice. In
control experiments about 18% of blood cells were detected to be Ly5.2
positive (Fig. 8B). No
Ly5.2+ cells could be detected in the peripheral blood of
recipients that received
Anxa5-lacZ+/Ly5.2+ PVCs
(Fig. 8C). Therefore, purified
Anxa5-lacZ+ PVCs do not exhibit the capacity to
reconstitute cells of the hematopoietic system in vivo.

Purified PVCs participate in muscle regeneration in vivo. Isolated
Anxa5-lacZ+ PVCs of adult brain meninges were injected
into regenerating areas of crushed tibialis anterior (TA) muscle of
immunodeficient NOD/SCID mice. By day 13, consecutive muscle sections were
analyzed for β-galactosidase activity by X-gal staining and expression of
desmin (B,E), PECAM (C) as well as CD34 (F) by immunohistochemistry. Injected
Anxa5-lacZ+ cells were detected at day 13 in the
zone of regeneration (arrows, A,D) in columnar structures associated with
intact desmin-positive muscle fibers (B,E). No staining was seen in intact
fibers. lacZ-expressing cells were found in areas positive for
CD34+ (D,F) but not in areas positive for PECAM (A,C). Scale bars:
50 μm.

Discussion

Perivascular cells (PVCs) are essential for the development of functional
vessels and contribute to the structural integrity as well as contractility of
vessels. Nevertheless, this cell population is poorly characterized and the
developmental origin of PVCs still remains unclear
(Gerhardt and Betsholtz,
2003). In this paper, we have isolated and characterized murine
Anxa5-lacZ+ PVCs, showing that these cells retain
the capacity to differentiate into adipocytes and osteoblastic cells but not
into hematopoietic lineages.

It has been suggested that pericytes and vSMC are phenotypic variants of a
continuous population of PVCs and have the potential to give rise to each
other, but may differentiate to chondrocytes, osteoblasts and adipocytes
(Canfield et al., 2000;
Nehls and Drenckhahn, 1993).
The lack of adequate markers during early development of vasculature causes a
major problem for analyzing the role of PVCs in this process. During growth,
remodeling and differentiation of the vasculature, expression of specific
markers like SMA and NG2-proteoglycan become indicative for PVCs only at later
stages (Ozerdem et al., 2001).
Three lines of evidence now clearly indicate that Anxa5-lacZ
expression represents a novel and highly specific marker for these PVCs during
development and in the adult. First, Anxa5-lacZ+
cells are exclusively detected in close proximity to PECAM-positive
endothelial cells and show colocalization with the markers SMA or NG2 in many
cells. Second, electron microscopy detects deposits of the
lacZ-product in cells directly contacting the endothelium. This can
be seen even in early embryos, where the characteristic common basement
membrane is not yet developed, as well as in pericytes of adult blood vessels
defined by their typical basement membrane
(Fig. 3). The third and most
important argument is seen with the expression analysis of isolated
Anxa5-lacZ+ cells. As previously shown, the
presence of NG2, SMA and PDFGRβ is highly indicative for pericytes or
pericyte-related cell populations
(Gerhardt and Betsholtz,
2003). All these markers are also significantly expressed in
purified Anxa5-lacZ+ cells from various sources.
The expression profile of these markers was identical among populations
isolated from E10.5, E16.5 and even adult meninges, which defines a common
conserved expression pattern at different stages of development. These data
clearly show that Anxa5-lacZ+ expression
correlate with typical markers of PVCs, even at very early stages of
development.

Purified Anxa5-lacZ+ PVCs do not have
hematopoietic capacity in vivo. Purified
Anxa5-lacZ+ PVCs from adult brain meninges
(Ly5.2+, 1 ×105 cells) were mixed with bone marrow
cells (Ly5.1+, 9 ×105) and injected intravenously
into recipient mice (Ly5.1+) after lethal irradiation. After 28
days, peripheral blood of recipients was analyzed by flow cytometry for the
presence of Ly5-markers. (A) No Ly5.2+ cells were detectable in
blood of the negative control mice, represented by non irradiated wild-type
Ly5.1+ mice without transplantation. (B) In the positive control,
18% of the blood cells derived from the injected Ly5.2+ BMCs. (C)
No Ly5.2+ cells could be detected after administration of
Anxa5-lacZ+/Ly5.2+ donor PVCs.

Recently, a transgenic mouse line (XlacZ4) was described to display
specific β-galactosidase expression in pericytes
(Tidhar et al., 2001). Yet it
is not clear which gene is reflected by the XlacZ4 gene-trap. Both
the XlacZ4 and Anxa5-lacZ reporters define overlapping
expression patterns, as they are expressed in PVCs of adult vasculature. It is
remarkable that both reporters are not expressed in the microvasculature of
the liver, highlighting the specific nature of hepatic microvasculature,
especially of the pericyte-related Ito cells
(Tidhar et al., 2001).
Nevertheless, there are significant differences early in development. Whereas
the XlacZ4 reporter is first active in the basilar artery within the head
mesenchyme of E10-E10.5 embryos, Anxa5-lacZ is already
expressed in angioblasts of the primary vascular plexus. At later stages, the
Anxa5-lacZ reporter is active in mesenchymal condensates
giving rise to chondrogenic and osteogenic tissues, whereas XlacZ4 is
expressed only in a subpopulation of cartilage cells in joints of the
appendages. Therefore, the expression of both reporter genes is similar at
later developmental stages, but Anxa5-lacZ expression may
define a unique population of vasculature-associated cells also early in
development.

During vasculogenesis, the expression of the Anxa5-lacZ
reporter gene was detected in cells forming the primary capillary plexus in
the yolk sac of E7.5 to E8.5 embryos. This staining resembles the angioblast
appearing at onset of vasculogenesis. This expression is reflected by Flk1
expression in the purified Anxa5-lacZ+ cells (not shown),
representing a characteristic marker of angioblasts of the yolk sac mesoderm
(Shalaby et al., 1995). Later
during angiogenic remodeling, expression of the Anxa5-lacZ
marker defines the population of PVCs. Many studies have indicated that PVCs
are recruited from stromal cells by mutual contacts with endothelial cells
(reviewed by Gerhardt and Betsholtz,
2003). Alternatively, trans-differentiation from endothelial cells
was discussed to contribute to the PVC cell pool. Flk1+ cells were
also identified in ES-cell cultures, which can serve as a progenitor for
endothelial cells and SMCs in vitro and in vivo
(Ema et al., 2003;
Yamashita et al., 2000).
Therefore, we assume that Anxa5 expression may represent a novel marker for a
subset of the mesenchymal stem cell compartment
(Dennis and Charbord, 2002)
that differentiates into angioblast and later strictly correlates with the
transition to PVCs. Although `it takes two to make blood vessels –
endothelial cells and pericytes' (Gerhardt
and Betsholtz, 2003), the understanding of PVCs is rather limited
in comparison to endothelial cells. Therefore, the purification of
Anxa5-lacZ+ PVCs represents a novel and unique
tool with which to characterize this cell type by in vitro and in vivo studies
in the mouse system.

The isolated PVCs clearly reflect a pericyte-like phenotype that is
indicated by their morphology, by the detection of NG2 and SMA protein in
cultured cells, and by their differentiation into adipogenic, early
chondrogenic and osteogenic cells. Earlier studies have indicated that bovine
derived pericytes, mesenchymal stem cells and adult trabecular-derived bone
cells are able to differentiate into chondrogenic cells and adipocytes
(Canfield et al., 2000;
Nuttall et al., 1998).
Interestingly, isolated cell populations were found to express different stem
cell markers, such as Flk1, Kit, Sca1 and CD34 (see
Fig. 5). This pattern reflects
the phenotype of aorta-derived multipotent progenitors
(Minasi et al., 2002), as both
pools express the `hemangioblast' markers CD34, Kit and Flk1. Additionally,
the stem cell marker Sca1 is expressed. It has been discussed that
Sca1+-expression in muscle defines a vascular-associated pool of
adult stem cells in skeletal muscle with a high degree of phenotypic
plasticity (Asakura et al.,
2002; Cao et al.,
2003; De Angelis et al.,
1999; Tamaki et al.,
2002). Surprisingly, this marker was also detected in
Anxa5-lacZ+ PVCs from early embryos and was even
upregulated in adult meninges. Moreover, Flk1 and VE-cadherin are expressed in
purified PVCs, two markers also detected in embryoid body-derived precursor
blast colonies of the hematopoietic as well as the endothelial cell lineage
(Kennedy et al., 1997).
Interestingly, the progeny of cultured ES-cells with hemangioblast potential
also express Flk1 and VE-cadherin, but not CD45
(Nishikawa et al., 1998).

In vivo regeneration experiments of muscle clearly show that isolated PVCs
are specifically incorporated into columnar structures associated with blood
vessels located between muscle fibers, but not integrated into newly formed
muscle fibers. This correlates with the finding that
Anxa5-lacZ+ cells do not express the
characteristic marker Pax7 for satellite cells, the major myogenic stem cells
(Seale et al., 2000) cannot be
detected. Therefore, Anxa5-lacZ+ PVCs may not
retain myogenic differentiation capacity in vivo. Nevertheless, we cannot
exclude the possibility that Anxa5-lacZ reporter expression
is inactivated in differentiated muscle fibers, as we never detected
expression of the reporter gene in skeletal muscle cells.

Multiple stem cell populations have been defined in skeletal muscle. The
side population (SP) represents a well characterized cell pool
(Goodell et al., 1997), which
is found associated to the vasculature and displays hematopoietic potential in
vitro (Asakura et al., 2002).
Muscle adult stem cells also exhibit hematopoietic capacity in vivo
(Gussoni et al., 1999;
Howell et al., 2002;
Jackson et al., 1999). The
markers CD34 and Sca1 are characteristically expressed by the SP cells
(Goodell et al., 1997),
although the correlation of CD34 expression and stem cell potential still
remains unclear (Parmar et al.,
2003). Sca1 expression is used as a marker for potential
hematopoietic cells between muscle fibers
(Asakura et al., 2002).
Although these markers are expressed in
Anxa5-lacZ+ PVCs and injected PVCs were found in
Sca-1+/CD34+ areas in regenerating muscle, we could not
define a capacity for hematopoietic differentiation of
Anxa5-lacZ+ PVCs isolated from adult meninges by
bone marrow reconstitution experiments.

Interestingly, the Anxa5-lacZ reporter is highly active
in mesenchymal condensations at E13.5, preceding formation of cartilage and
bone during development, but also later in all differentiated skeletal
elements (Brachvogel et al.,
2001). Additionally, isolated PVCs had the potential to develop
into early chondrogenic, osteogenic and adipogenic lineages. Recently, it was
shown that the embryonic dorsal aorta of E10.5 embryos contains progenitor
cells termed meso-angioblast, which are able to participate in the development
of perichondrium and cartilage, connective tissue, smooth muscle and cardiac
muscle (Minasi et al., 2002).
It became obvious that a mesenchymal stem cell compartment may exist that is
represented by a spectrum of related cells with capacity for phenotypic
differentiation into stroma cells, adipocytes, chondrocytes or bone cells
(Caplan, 1991;
Dennis and Charbord, 2002).
This model differs from the standard paradigm that postulates hierarchical
lineages during development, as in the hematopoietic system, by a high degree
of plasticity during lineage progression and transition
(Theise et al., 2003). We
propose that Anxa5-lacZ expression defines a subset of the
mesenchymal stem cell compartment and may reflect some aspects of various
differentiation pathways during development.

In this paper, we describe for the first time the use of Anxa5 expression
for the isolation of a novel cell population resembling perivascular cells
(pericytes and vSMCs), from mouse tissues, characterized by the expression of
pericyte-specific markers. In future, this unique method will help to define
the capacities of the poorly characterized PVCs. Our data show that these
cells additionally display markers of mesenchymal stem cells and have the
capacity to differentiate into adipocytes and osteoblastic cells. Therefore,
expression of Anxa5 defines a novel subset of the mesenchymal stem cell
compartment that reflects variable lineage progression and phenotypic
plasticity.

Acknowledgments

This project was supported by Deutsche Forschungsgemeinschaft (Po340/4).
Cell sorting was kindly performed by Peter Rower (Erlangen). Antibodies for
collagen VI were kindly provided by R. Timpl (Martinsried; deceased 2003) and
antibodies for collagen IX were generously provided by Vic Duance
(Cardiff).